The Vertebrate Embryology Section seeks to elucidate molecular and cellular events that drive gastrulation. During vertebrate gastrulation, embryos undergo dramatic morphogenesis, beginning as a simple assembly of homogenous cells, and emerging as highly diversified beings with a recognizable vertebrate body plan. Errors during gastrulation, due to genetic mutations or environmental insults, can lead to developmental deformities or miscarriage. A more precise understanding of gastrulation will enable the development of diagnostic and therapeutic tools for human developmental disorders.[unreadable] [unreadable] We have three major projects focused on elucidating mechanisms of gastrulation. [unreadable] [unreadable] Project 1. Defining upstream and downstream components of Nodal signaling in gastrulation. Modulators of sqt penetrance. cyc embryos have a more limited mesoderm and endoderm deficit than sqt;cyc double-mutant embryos, namely a reduction in anterior axial mesoderm and endoderm, leading to holoprosencephaly (HPE). This phenotype is seen in only a fraction of sqt mutants, with many appearing wild type. Here at NHGRI, we examined the basis of sqt phenotypic variability and found that co-inherited genes, environmental temperature and Heat Shock Protein 90 function all influence HPE incidence in these mutants. [unreadable] Function of FoxH1. One challenge to dissecting the Nodal signaling pathway is the diversity of phenotypes resulting from perturbations of FoxH1 function. At NHGRI, we have characterized zebrafish embryos treated with antisense morpholino oligonucleotides (MOs) designed to block translation of maternal and zygotic FoxH1 protein. These embryos display a striking down regulation of keratin genes and dramatic disruptions in cell movement. This phenotype, similar to antisense FoxH1 disruptions in Xenopus laevis, suggests that FoxH1 has Nodal-independent functions. By contrast, a milder phenotyope is seen in the zebrafish schmalspur (sur) mutants, two FoxH1 alleles with point mutations in the winged-helix DNA binding domain. We have accordingly hypothesized that the severe phenotype associated with FoxH1 knockdown is due to the loss of FoxH1 functions outside of the DNA-binding domain.[unreadable] [unreadable] Project 2. [unreadable] [unreadable] Identifying roles of RhoGTPase signaling in gastrulation movements. An attraction of zebrafish embryology is its amenability to time-lapse visualization of morphogenesis. The standard system for time-lapse documentation is invasive and not well suited for parallel examination of multiple specimens. In order to efficiently screen morphogenetic phenotypes resulting from dozens of different injected MOs targeting an entire gene family like the RhoGEFs, we designed, with help from NIH?s Mechanical Instrument Design and Fabrication Branch, a non-invasive holding system that allows for the parallel time-lapse documention of 54 embryos. With assistance from NHGRI?s Bioinformatics and Scientific Programming Core, we compiled a list of 71 known and predicted RhoGEF genes and determined by RT-PCR that 48 are expressed during the first 12 hours of development (cleavage, blastula and gastrula stages). We injected individual MOs targeting 23 of these expressed RhoGEFs and documented their effects on early morphogenesis (6-24 hpf). To avoid false positive results, we designed second non-overlapping MOs against all RhoGEFs for which an initial MO had produced a phenotype. We thus identified five RhoGEFs for which two independent MOs produced the same phenotype. Three of these, homologues of ARHGEF16, Frabin and Net1, respectively, disrupted epiboly. Two others, ARHGEF10 and PLEKHG4 homologues, caused post-gastrulation defects during somitogenesis stages. [unreadable] Epiboly is a form of epithelial sheet migration and elucidating its details may inform our understanding of other epithelial sheet movements such as those of human wound healing and eyelid development. We have suspended our RhoGEF screen in order to characterize the three RhoGEFs affecting epiboly. Interestingly, the MO-induced phenotypes corresponding to the Frabin and Net1 homologues are indistinguishable and co-injection of these MOs fails to produce exacerbated defects. This suggests that each of these RhoGEFs is an essential component of the same process. At the same time, published literature indicates that Frabin homologues target CDC42, while Net1 homologues target Rho. Thus Frabin and Net1 may impinge on a single biological process via the regulation of distinct RhoGTPases.[unreadable] [unreadable] Project 3. Defining roles of blastula margin-enriched proteins and behaviors of blastula margin cells. Classical embryology has been epitomized by elegant experiments requiring precise microdissection. The power of these approaches and the artistry of certain researchers is the stuff of legend. There are nonetheless challenges where an even higher degree of surgical precision, reproducibility and flexibility are desirable. A precise separation of newly-specified germ layer precursors in zebrafish embryos, for instance, is unimaginable using conventional tools. Part of the excitement of being an NHGRI investigator is our mandate to engage in high-risk research. Accordingly, I have expended a portion of my resources developing a novel technique for precisely microdissecting embryonic regions of interest: FACS-assisted microdissection of photolabeled cells (FAM-P). For our first FAM-P study, we focused on separating marginal mesoderm and endoderm precursors from neighboring ectodermal precursor cells, using the two fluorescent states of Kaede, a photoconvertible protein isolated from stony coral. We injected purified Kaede into embryos and allowed them to develop to the late blastula stage (5 hpf). We then used a scanning laser confocal microscope to selectively photoconvert Kaede from green to red fluorescence using a 405 nm laser along 4-6 tiers of blastula margin cells, comprising most mesoderm and endoderm precursors. Labeled embryonic cells were dissociated and, with help from NHGRI?s Flow Cytometry Core, subjected to fluorescence-activated cell sorting (FACS). Preliminary studies indicate that a reasonable number of the cells thus sorted are healthy and can be cultured overnight. To examine gene expression differences between the germ layer precursor populations, RNA was extracted from sorted cells, linearly amplified, labeled and hybridized to a custom microarray, provided by NHGRI?s Genomics Core, with oligo representation of more than 20,000 unique zebrafish genes. In validation of our strategy and demonstrating that the sorted cells have maintained their cellular identity, many genes known to have elevated expression in the late blastula margin were re-identified in margin cell-derived RNA, including no tail, fgf8, chordin, wnt11, cyclops and squint. Furthermore, we found 47 genes with a greater than 3-fold enrichment in the mesendoderm precursor population (p < 0.0015) whose late blastula expression has not been previously described, and a similar number of significantly-enriched ESTs. WISH studies have validated the margin-specific expression of ten of these genes (out of 21 tested).